Genomic monitoring of SARS‐CoV‐2 variants using sentinel SARI hospital surveillance

Abstract Background To support the COVID‐19 pandemic response, many countries, including Belgium, implemented baseline genomic surveillance (BGS) programs aiming to early detect and characterize new SARS‐CoV‐2 variants. In parallel, Belgium maintained a sentinel network of six hospitals that samples patients with severe acute respiratory infections (SARI) and integrated SARS‐CoV‐2 detection within a broader range of respiratory pathogens. We evaluate the ability of the SARI surveillance to monitor general trends and early signals of viral genetic evolution of SARS‐CoV‐2 and compare it with the BGS as a reference model. Methods Nine‐hundred twenty‐five SARS‐CoV‐2 positive samples from patients fulfilling the Belgian SARI definition between January 2020 and December 2022 were sequenced using the ARTIC Network amplicon tiling approach on a MinION platform. Weekly variant of concern (VOC) proportions and types were compared to those that were circulating between 2021 and 2022, using 96,251 sequences of the BGS. Results SARI surveillance allowed timely detection of the Omicron (BA.1, BA.2, BA.4, and BA.5) and Delta (B.1.617.2) VOCs, with no to 2 weeks delay according to the start of their epidemic growth in the Belgian population. First detection of VOCs B.1.351 and P.1 took longer, but these remained minor in Belgium. Omicron BA.3 was never detected in SARI surveillance. Timeliness could not be evaluated for B.1.1.7, being already major at the start of the study period. Conclusions Genomic surveillance of SARS‐CoV‐2 using SARI sentinel surveillance has proven to accurately reflect VOCs detected in the population and provides a cost‐effective solution for long‐term genomic monitoring of circulating respiratory viruses.


| INTRODUCTION
Since the start of the SARS-CoV-2 pandemic in 2019, genomic surveillance to detect emerging variants of concern (VOCs) and variants of interest (VOIs) has been widely used, especially to guide public health actions, initiate early characterization of emerging variants, understand the spatio-temporal spread of the virus, and understand the impact of emerging mutations on treatment efficacy. [1][2][3][4][5][6][7][8][9] To date, the World Health Organization (WHO) has designated five VOCs worldwide after the start of the pandemic with the original Wuhan strain. [10][11][12] The SARS-CoV-2 Alpha variant (B.1.1.7) was first identified in the United Kingdom (UK) in late summer to early autumn 2020 and caused a rapid increase in COVID-19 cases in the UK by the end of 2020. 13,14 During the same period, a rapid resurgence of the epidemic was caused by the Beta (B.1.351) variant in South Africa, [15][16][17] and in Brazil, the emergence of a novel VOC, referred to as Gamma (P.1), was reported around early November 2020. 16 19 During the acute pandemic phase, it has been considered essential to detect, monitor, and assess virus variants that can result in increased transmissibility and disease severity or have other adverse effects on public health and social control measures. 2,20 To obtain timely and accurate information on the emergence and circulation of VOCs and VOIs, robust surveillance systems, including a well-defined sampling and sequencing strategy, were required and were implemented in many countries. 21 It is within this context that the European Center for Disease Prevention and Control (ECDC) recommended two complementary sampling approaches. First, a representative sampling of SARS-CoV-2 RT-PCR positive cases from existing populationbased surveillance systems and, second, a targeted sampling of SARS-CoV-2 positive cases occurring in special settings or populations. 2 In order to comply with these recommendations, Belgium set up a national baseline genomic surveillance (BGS) aiming at sequencing a representative subset (5-10%) of all SARS-CoV-2 PCR positive samples in order to follow-up trends for the circulating viruses and to detect emerging variants when they reach a certain proportion, typically around 1%. 2,[22][23][24][25] Additionally, an active genomic surveillance was also set up to target specific indications, including unusual outbreaks, persisting infections in immunocompromised patients, and returning travelers from a zone at risk. 23,24,26 Patients hospitalized with severe acute respiratory infections (SARI) were at first not included in these indications, although being a very important group to evaluate the impact of SARS-CoV-2 on human health. [27][28][29][30] Sentinel surveillance networks have been operating for influenza virus for many years. [27][28][29]31,32 In Belgium, SARI surveillance exists since 2012 and consists presently of a network of six hospitals sending respiratory samples and clinical information from patients fulfilling the case definition to the National Influenza Centre. In particular, SARI surveillance has proven to be very useful to evaluate the severity of infections caused by different respiratory viruses, including influenza virus. [27][28][29][30][33][34][35][36][37] Our group also demonstrated that non-influenza respiratory viruses (NIRV) have an important contribution to the burden of SARI, with overall one third of the SARI cases showing positivity for one or more of the respiratory viruses tested (i.e., coronavirus, human metapneumovirus, rhinovirus, enterovirus, and parainfluenza virus or respiratory syncytial virus). [27][28][29] The information retrieved from the Belgian SARI network system did not only allow to evaluate the severity of these respiratory viruses against the burden of influenza but also to investigate the vaccine efficacy and epidemiology of these viruses. Additionally, the provided data were considered to be beneficial for clinical management of SARI patients. [27][28][29] In this study, we evaluated the performance of a SARI-based SARS-CoV-2 genomic surveillance for the five different VOCs based on the Belgian sentinel hospital network during the pandemic. This evaluation was performed based on different criteria, namely, the ability to correctly follow major trends obtained from the exhaustive baseline genomic surveillance, the ability to promptly detect major introduction events, and the ability to detect minority variant populations.
After more than 2 years of crisis due to the SARS-CoV-2 pandemic, and with many countries now entering the recovery phase and planning for long-term surveillance of respiratory pathogens, we demonstrate here that the SARI surveillance network is an appropriate and cost-effective tool to monitor the circulation of SARS-CoV-2 variants or variants of other respiratory pathogens.

| METHODS
Belgian sentinel SARI surveillance has been in place since 2012 and is composed of six hospitals spread over the whole country ( Figure 1, map created using the Free and Open Source QGIS, version 3.22.5). [27][28][29] The overall catchment population of the current network is estimated at 992,310 inhabitants, which corresponds to 8.6% of the Belgian population.
At these hospitals, respiratory samples (nasopharyngeal swabs, aspirates, or broncho-alveolar lavages) were taken from adults and children fulfilling the Belgian SARI definition (adapted from the WHO 2014 SARI case definition). A SARI case is defined as a person suffering from an acute respiratory illness with onset within the last 10 days of (1) history of fever or measured fever of ≥38 C, (2) cough or dyspnea, week. ISO weeks were used to perform this calculation. We defined the epidemic growth phase of a VOC as the period for which the exponential increase of its weekly reported proportion (%) is a constant during at least 4 weeks in the BGS, which was calculated using a linear regression curve. [42][43][44] The slope value a of the linear regression curve (equation format y = ax + b) within that time window represents the relationship between the detection levels (%) of each VOC in relation with time, and thus provides a measure of how quickly a VOC is growing. The R 2 value was used as a measure of the quality of the linear regression curve for the used dataset. (non-travel-related) detection of the virus in the general population.
Since then, out of 5,695 respiratory samples received between March 13, 2020, and December 31, 2022, 1,558 samples tested positive for SARS-CoV-2 by qPCR, and 1,103 (71%) had a Ct value that enabled sequencing. In total, 925 samples were sequenced successfully (Supporting Information S1), covering the different SARS-CoV-2 waves of the pandemic in Belgium. Each week, 1 to 44 SARS-CoV-2 positive SARI samples were sequenced, except for some weeks where none were sequenced. Only the second wave (August 2020 till February 2021) was missed due to a temporary disruption of the SARI surveillance system, which resumed in 2021-W04. Since the BGS started officially from 2021-W07, 859 sequences from the SARI surveillance were included in the analysis. Variant trends provided by these 859 sequences from the SARI surveillance were compared with those determined by the BGS including a total of 96,251 sequences from 2021-W07 until 2022-W52. Lineage analysis showed a similar pattern to that observed in the national baseline genomic surveillance ( Figure 2).
F I G U R E 2 SARS-CoV-2 VOC dynamics during the COVID-19 pandemic registered by the SARI surveillance (upper graph) and the national baseline genomic surveillance (lower graph). Both surveillance systems shows similar patterns. The national baseline genomic surveillance officially started in Week 7 of 2021, whereas SARI surveillance sequencing was retrospectively performed since 2020-W09. The results of the baseline genomic surveillance are based on a total of 96,251 sequences, whereas those of the SARI surveillance system are based on 964 sequences. Wuhan-like strains and non-VOC variants (detected in 2020 by SARI surveillance) are included in "other variants." The speed of epidemic growth was evaluated for each VOC using the slope values at the epidemic growth phase in the baseline geno- For this study, we defined a variant as emerging within the Belgian population based on the exponential properties (i.e., the slope and value for R 2 ) of its weekly frequency detection curve and its weekly calculated detection level within the total number of sequences as calculated from the baseline surveillance genomic data.
We have set empirically following criteria: R 2 > 0.95, slope > 5 and maximal weekly detection level >15%. The R 2 value of 0.95 is a general accepted value to define a reliable regression model. The slope value has been arbitrary chosen and set at five, as a proof-of-concept to express an important increase in VOC numbers (five times) per time unit (1 week). Where ECDC uses a threshold of 10% to indicate the start of the seasonal epidemic of influenza based on sentinel specimens, 45

| DISCUSSION
During the SARS-CoV-2 pandemic, many countries integrated SARS-CoV-2 surveillance in their existing sentinel surveillance systems successfully and allowed those countries to evaluate different epidemiological aspects of this new respiratory pathogen. 9,30,37,46,47 The Belgian National Influenza Centre also integrated SARS-CoV-2 detection in the existing hospital SARI surveillance for influenza. 29 This allowed sequencing of SARS-CoV-2 positive samples and thus week on, the SARI surveillance has been running continuously, all weeks of the year. Our results show that even with a relatively low number of samples and delay in timing of sequencing, SARI surveillance succeeded in detecting the minor and major VOCs that eventually emerged in Belgium in a timely manner. To be able to detect viral variants during both their endemic or pandemic circulation, a yearround SARI surveillance, subject however to some organizational improvements, should be envisaged and encouraged. This should ideally involve an increase of the number of the participating hospitals to improve the catchment population and being more representative for the whole country but should also require better resources for sampling and faster sample shipment. Not only pandemic preparedness and response will be strengthened, but also the public health management of the yearly epidemics of a number of respiratory viruses will benefit from this system. [27][28][29]56 The performance of the SARI surveillance system can be increased by including different pathogenspecific labs or institutions.
Besides the difference in sequencing strategy, an important difference between both surveillance systems lies in the sampling strategy itself. While the BGS collects SARS-CoV-2 positive samples from over 40 diagnostic laboratories, relies on decentralized sequencing, but centralized data repository and analysis, sampling in SARI surveillance is based on six sentinel sites recruiting samples based on the clinical presentation that is limited to patients that fulfill the SARI case definition. 26,28,57 In the context of SARI surveillance in Belgium, testing and sequencing are centralized in the public health laboratories of Sciensano. As the SARS-CoV-2 pandemic evolved, both natural immunity due to virus exposure and induced immunity from vaccination programs increased within the population and conferred protection against severe disease, and it will continue to do so in a post-pandemic era. 57,58 Together with the changing characteristics of variants while adapting to its host to evade immune response, SARS-CoV-2 will circulate in the general population without necessarily causing hospitalization to the same extent as observed during the SARS-CoV-2 pandemic, thereby limiting severe infections more to the high-risk groups (i.e., people aged >60 years or with underlying health conditions) related to this pathogen. [59][60][61][62] This could result in a lower detection of variants causing mild disease in the targeted population of the SARI surveillance, while variants causing more severe disease might be overrepresented. However, it will become less important to capture these variants causing mild disease in a timely manner, since specific measures are no longer put in place to protect the general population from these particular variants. Anyway, patients suffering from mild respiratory infections are well covered by the existing ILI (influenza-like illness) surveillance that involves a network of sentinel general practitioners in Belgium since 1979. 63 This ILI surveillance could complement the SARI surveillance network, the latter still focusing on variants causing severe respiratory infections.
Since both SARI and ILI surveillances sampling are symptom based, Genomic SARI surveillance shows timely detection for each VOC as compared to the baseline surveillance, except for BA.3 (omicron), which was not detected. The observed trends in the proportion of weekly positives of a variant in SARI surveillance are comparable to the waves observed in the baseline surveillance. they both have the advantage of lowering the risk in biased sample selection which at some times will be the case for the baseline surveillance, where samples with specific characteristics of variants of interest or concern might be prioritized for sequencing to rapidly detect introduction of a variant in the country. 26,28 Also, as the national testing indications change from testing all suspected COVID-19 patients to only testing very specific subgroups, the representativeness of a BGS will change over time, while the representativeness of the SARI surveillance remains the same.
Viruses evolve continuously and the BGS aims for the rapid detection of new introductions in the general population and emergence of new variants in near real-time, in order to inform the local authorities and allow a prompt risk assessment. 3,48 While immediate launch of biomedical research has shown to be efficient on multiple occasions, public health actions such as travel restrictions and targeted testing and tracing during the pandemic following the detection of a new variant have however not proven to allow a sustained containment. 9,11,12,26,64 Upon its first detection, a variant is often already circulating at low to moderate levels within the population, so often variant-specific measures have only limited effect on the further spread. 6,9,64,65 Also, within risk assessment, hazard assessment not only relies on the virological information of a variant retrieved from testing of viral isolates, but also on epidemiological information retrieved after a variant has been circulating for some time, thereby using the numbers of deaths and hospitalized cases that were registered. SARI surveillance aims to gather information on the circulating strains in human cases suffering from a severe respiratory infection during the endemic, epidemic and/or pandemic circulation of a virus. 32 Although SARI surveillance is less timely and sensitive compared to the baseline genomic surveillance in its current format, it gives a well-founded assessment on the disease impact of pathogen circulation in a country. This allows for monitoring of locally circulating viruses for antiviral sensitivity or strain identification for the vaccine composition for the upcoming season, both existing already for influenza and possibly applicable for SARS-CoV-2 and other pathogens as well. 9,[27][28][29]31,[66][67][68] As suggested by Brito et al., 21 a nationwide strategy allowing to sequence at least 0.5% of the positive cases, with a turnaround time (time in days between sample collection and genome submission) of less than 21 days, could be a benchmark for viral pathogens genomic surveillance efforts. This means that to maintain its sensitivity, a national BGS would require a high capacity to continuously sequence these important numbers of samples, and the organization of their transport to the sequencing centers to allow near real-time surveillance should be guaranteed as well. In a different approach, sentinel surveillance provides an alternative solution to measure the impact of a virus or virus variant in the population using a lower number of sequences. 9 Moreover, the SARI sampling strategy remains unaffected by changes in testing indications. Another major benefit of using SARI and ILI surveillance in genomic SARS-CoV-2 variant detection is the availability of clinical patient information allowing for further analysis on severity and/or vaccine effectiveness using the same data. The clinical information sent alongside with the samples has not been used in the current study since it is out of the scope of this publication. Besides, the respiratory specimens sampled in SARI and ILI surveillance undergo long-term storage and allow for retrospective analysis, as demonstrated in the current study.
We think that sentinel ILI and SARI surveillance networks constitute a useful tool to monitor the circulation of SARS-CoV-2 variants, or other respiratory pathogens, subject however to some organizational and logistical adjustments to achieve this purpose and as described in the ECDC guidelines. 31

| CONCLUSIONS
In conclusion, our results show that sentinel SARI surveillance can give a valuable reflection of the circulation of the different SARS-CoV-2 variants of concern during pandemic and endemic periods, especially for variants related to severe respiratory infections. Sentinel SARI surveillance may represent a relatively cost-effective and sustainable tool to contribute to genomic surveillance, including in lowand middle-income countries.
To reflect circulation of variants causing mild disease in the general population, complementary analysis of ILI surveillance samples from a network of general practitioners could be considered, which is in line with the ECDC recommendation to use existing, sentinel surveillance systems for genomic surveillance of viral pathogens.
Improved and scalable genomic sentinel surveillance systems at a national level, such as integrated ILI and SARI surveillance networks for respiratory pathogens, should be considered as a valuable and reliable option to strengthen the pandemic preparedness and response.